1. Field of the Invention
The present application relates generally to the field of reagents, synthesis and purification of oligonucleotides.
2. Background Art
Oligonucleotides are short nucleic acid polymers, typically containing from a few to several hundred nucleotides. They are important tools for genomic research and biotechnology. Although oligonucleotides can be generated by cleavage of longer precursors, they are now more commonly synthesized from monomers in a sequence-specific manner. They are typically synthesized on solid support using phosphoramidite chemistry or phosphonate chemistry.
FIG. 1A shows a typical synthetic cycle for solid phase oligonucleotide synthesis using phosphoramidites. The four major steps include: 1) Detritylation; 2) Coupling; 3) Capping; and 4) oxidation. As shown in FIG. 1, a first nucleotide is chemically bonded to a solid support (e.g., controlled pore glass beads). These can be purchased from commercial sources with the first nucleotide pre-coupled to the solid support.
To start the synthesis, the 5′-hydroxy protecting group (commonly used 5′-hydroxyl protection group is a trityl (i.e., —C(Ph)3) or a 4,4′-dimethoxytrityl group, which may be abbreviated as DMT, DMTr) on the first nucleotide is removed using a mild acid (e.g., 2 or 3% trichloroacetic acid in an inert solvent, such as dichloromethane or toluene). Then, a 5′-DMTr-protected second nucleotide having a 3′-phosphoramidite group (e.g., 3′-O-(2-cyanoethyl-N—N-diisopropyl)phosphoramidite) is coupled to the free 5′-hydroxyl group on the first nucleotide on the solid support. The coupling can be achieved with activating the phosphoramidite using an acidic azole catalyst (e.g., 1H-tetrazole) in acetonitrile. The activated phosphoramidite then reacts with the free 5′-hydroxyl group to form a phosphite triester linkage.
Because the coupling reaction is never 100% efficient, some free 5′-hydroxyl group would remain. Any non-reacted 5′-hydroxyl group is capped with acetic anhydride and an acylation catalyst (e.g., N-methyl imidazole) to prevent the failure sequences from reacting in the next cycle. After capping, the phosphite triester is oxidized to convert it to a phosphate linkage. Oxidation can be accomplished with iodine in the presence of a weak base (e.g., pyridine). These steps complete the first coupling cycle. The processes can be repeated many cycles until the desired oligonucleotide is synthesized.
A similar method is illustrated in FIG. 1B, which uses H-phosphonate monomers instead of phosphoramidites. Most steps are similar, except that only one oxidation step is needed and is performed at the end of the synthesis.
At the completion of the chain elongation, the oligonucleotide is still attached to the solid support and is fully protected. To furnish a functional oligonucleotide, the protecting groups need to be removed and the oligonucleotide product needs to be released from the solid support. These can be accomplished using a base solution (e.g., ammonium hydroxide or aqueous methylamine) and perhaps other pretreatment steps (e.g., deprotection of 2-cyanoethyl protection group).
The above-described synthesis procedures are applicable for the synthesis of DNA or RNA oligomers. However, for RNA synthesis, the 2′-hydroxyl groups need to be protected with groups that can survive the reactions conditions. Typical 2′-hydroxy protecting groups include t-butyldimethylsilyl (TBDMS) and triisopropylsilyloxymethyl (TOM).
In general, the coupling step is never 100% complete. Incomplete reaction leads to the formation of short-mers. As noted above, the short-mers are typically capped off with a reagent, such as acetic anhydride, to prevent them from growing during iterative solid phase synthesis. This generally results in a pool of oligomers consisting of short-mers (n−1, n−2, n−3, etc. mers) and the desired full-length product. FIGS. 2A and 2B show an HPLC chromatogram and PAGE analysis, respectively, to illustrate the fact that the product is a mixture containing some failure sequences.
The products from oligonucleotide synthesis always contain failure sequences (shorter oligonucleotides, mostly n−1 mers). Traditionally, these crude products are purified using HPLC or gel electrophoresis, such as PAGE. Though these methods are acceptable for the purification of short oligonucleotides, they are unacceptable for the purification of longer oligonucleotides (e.g., 50-300-mers). In addition, these methods can be expensive and time consuming, can consume vast amounts of solvents, and can be limited to the use of trained professionals.
To facilitate the synthesis and purification of oligonucleotides, alternative approaches have been investigated, such as using synthetic purification handles to circumvent the use of HPLC or PAGE. Examples of these alternative methods include fluorous-affinity extraction (Beller, C.; Bannwarth, W., Noncovalent Attachment of Nucleotides by Fluorous-Fluorous Interactions: Application to a simple Purification Principle for Synthetic DNA Fragments, Helv. Chim. Acfa 2005, 88, pp. 171-179; WO 2006/081035 A2), biotin-avidin enabled affinity extraction (Fang, S.; Bergstrom, D. E., Reversible Biotinylation of the 5′-Terminus of Oligodeoxyribonucleotides and its Application in Affinity Purification, Current Protocols in Nucleic Acid Chemistry, John Wiley & Sons, Inc. 2001; Fang, S.; Bergstrom, D. E., Reversible 5′-end Biotinylation and Affinity Purification of Synthetic RNA, Tetrahedron Letters 2004, 45 (43), 7987-7990), and reaction-based extraction methods via Diels-Alder (WO 2003/062452 A2) and polymerization (U.S. Patent Publication No. 2008/0081902 A1).
The affinity-based methods (i.e., fluorous affinity extraction and biotin-avidin methods) require complex synthesis to generate the tagged-oligonucleotide handles, which can be costly. In the case of fluorous-affinity extraction, the fluorous-containing phosphorylating reagent couples too slowly to be used as a capping agent for long oligonucleotide synthesis. For use as the last monomer, the 5′-DMTr fluorous-modified phosphoramidite monomers are sparingly soluble in acetonitrile, and couple slowly and inefficiently. They also require a final acidic treatment, which may cause depurination and affect the integrity of long oligonucleotides. This strategy also requires a fluorous phase affinity column, which increases complexity and cost, and may not be amenable to high throughput or large scale purification.
Biotin-avidin enabled affinity extraction has not been widely used and has never been used as a capping reagent to trap failure sequences. The biotin phosphorylating reagents, or 5′-modified phosphoramidite monomers, are expensive to manufacture due to the cost of biotin itself. In addition, the biotin phosphoramidites (phosphorylating reagents and monomers) are not very soluble in acetonitrile and the recommended coupling times are slow (i.e., about 15 min). Thus, they are not useful as capping reagents. In addition, this method has not been shown to be useful for long oligonucleotides. Also, this method relies on biotin's affinity to streptavidin-beads, which are expensive in a high throughput or large scale platform.
Due to the costs and other issues with the affinity-based approach, alternative methods that have been examined, such as reaction-based methods. These methods include those using Diels-Alder and polymerization. In the Diels-Alder approach, a diene-phosphorylating reagent is used to cap the truncated sequences, which can then be later pulled out of the product mixtures via a 4+2 cycloaddition with a maleimide-containing solid support. In practice, this method is restricted to the purification of short oligonucleotides (less than 20-mer) and the final yield and purity is poor due to inefficient reactions.
The polymerization method uses an acrylamide phosphorylating reagent to cap off the truncated (failure) sequences. Upon completion of the synthesis, radical polymerization is initiated to form polymers, which can then be separated from the full-length product. Acrylamide phosphoramidite monomers have also been used as the last nucleotide in a chain and the full-length products can be trapped by radical polymerization, thereby separating them from the short-mers, which do not contain an acrylamide moiety. Again, this method is practical for short oligonucleotides, e.g., less than 20-mers, and there is concern that radical polymerization may alter the integrity of the desired oligonucleotide due to side-reactions. Furthermore, the overall purity and recovery of the desired full-length products are less than optimal. Therefore, this method may not be suitable for high throughput purification or large scale production.
While these prior art methods are useful in some situations, there remains a need for better methods for the synthesis and purification of oligonucleotides.